The lack of knowledge on the biosynthetic pathway leading to threonylcarbamoyladenosine (t6A) has led to a fundamental gap in understanding the role this universal modification of the tRNA anticodon stem loop (ASL). As with other modifications of the ASL, t6A is expected to have critical roles in recognition of tRNAs by the specific components of the translation apparatus such as aminoacyl tRNA synthetases and cognate mRNAs. The long-term goal of our research is to pursue fundamental knowledge on the function of complex tRNA modifications and the enzymes required their biosynthesis, and elucidate their roles in core cellular processes. The current application focuses on t6A with an emphasis on contrasting the eukaryotic and prokaryotic biosynthesis pathways. The central hypothesis is that t6A enzymes are critical for both translation and DNA maintenance. This hypothesis derives from the recent discovery of two t6A biosynthetic enzymes by the applicant's laboratory: the Sua5/YrdC and the Kae1/YgjD families. Preliminary studies show that while t6A is important for optimal cell growth in yeast, the two genes yrdC and ygjD are absolutely essential in bacteria, suggesting that t6A is required for prokaryotic life. In addition, studies by other laboratories have shown that both families are linked to telomere maintenance in yeast and that the YgjD family is involved in maintenance of DNA integrity in bacteria and mitochondria. The rationale for the proposed research is that, once the roles of these gene families in translation and DNA maintenance are understood, the reasons for their essentiality in prokaryotes and their importance in telomere maintenance will become apparent, and together this knowledge will open the door to novel applications in the fields of antibiotics and anticancer targets. This central hypothesis will be tested by pursuing three specific aims: 1) Decipher the complete t6A biosynthetic pathway; 2) Determine the role of t6A in translation in vivo; and 3) Determine if t6A has cellular functions not directly linked to translation. Under the first aim, candidate enzymes have been expressed and substrate RNAs transcribed. Under the second aim, several dual reporter plasmids that allow testing the role t6A in initiation and frame maintenance have been constructed along with tRNA microarrays to test the effect of t6A on aminoacylation. Under the third aim, conditional essential E. coli strains that express yrdC or ygjD under the PTet promoter have been constructed and will be used in suppressor screens and strategies to identify other targets for modification by t6A have been developed. The approach is innovative because comparative genomic methods were used to guide the experimental effort, and this work is revealing new links between tRNA modification and DNA maintenance. The proposed research is significant because it will advance our understanding of the role of this critical tRNA modification in fundamental cellular events and could reveal new core regulatory mechanisms. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the elucidation of a universal tRNA modification pathway essential for accurate protein translation in all kingdoms of life, and for telomere maintenance in eukaryotes, is ultimately expected to reveal novel antibacterial and anticancer targets. Thus, the proposed research is relevant to NIH's mission to support research that increases understanding of life processes and lays the foundation for advances in cancer and/or antibacterial therapeutics.